Neural crest (NC) is a transient embryonic multipotent group of cells that arises within the dorsal neural tube, to forms cartilage and bone of the craniofacial skeleton, among other derivatives in vertebrate embryos. Therefore, craniofacial abnormalities are usually attributed to problems in neural crest cell development and depending of which phase of neural crest cell development is disrupted, very different craniofacial anomalies can manifest. Epithelial-to-mesenchymal transition (EMT) is one of the first events before initiation of the bona fide neural crest program, and the disruption of this step can result in very severe craniofacial anomalies. EMT is accompanied by changes in expression of members of the cadherin family molecules, including the down-regulation of Cad6B prior NC delamination. This process is regulated by Snail family members, which directly bind the Cad6B promoter and repress its transcription. However, the mechanisms underlying the role of repressive and activating signals in EMT are likely to be complex and involve multiple and interconnected factors. Here, we explore the possible role of epigenetic modification in this process and specifically the role of PHD12 (named for homology to plant homeodomain 12), a gene discovered as upregulated during neural crest induction, in cooperation with Snail2. Different studies have demonstrated that both Snail and PHD12 can recruit Sin3A/HDAC complex. This large multiprotein complex is able to deacetylate the histones located on the proximity of promoters to repress the target gene. Although Snail2 can bind to the E-boxes of Cad6B, we hypothesize it requires a partner able to read the epigenetic marks and recruit the repressive complex Sin3A. The goal of this application is to demonstrate that the direct interaction between Snail2 and PHD12 makes possible to recruit the repressive complex Sin3A/HDAC to complete shutdown Cad6B expression via histone deacetylation. This application involves three specific aims that include: Aim 1, characterize the role of PHD12 in NC EMT. Our preliminary data show that PHD12 is expressed in a time and location appropriate to be associated with NC prior their delamination. We hypothesize and will test whether the presence of PHD12 affects the NC EMT related genes. In the Specific Aim 2, we will determine the mechanistic analysis of Snail2, PHD12 and Sin3A interaction, and their role in Cad6B repression. Our preliminary ChIP experiments provide evidence that PHD12 interacts with Cad6B locus. We hypothesize and will test the interaction between Snail2-PHD12-Sin3A as well as the requirement of this interaction to bind to the Cad6B locus to repress it via promoter deacetylation. Finally, in the Specific Aim 3 will determine the role of PHD12 in the migration of NC cells and the formation of craniofacial derivatives. We will focus on the role of PHD12 in long term effects on neural crest migration to the branchial arches and subsequent differentiation. To extend this to later times when neural crest cells are forming critical craniofacial derivatives, chick embryos will be allowed to develop in ovo to later stages (e.g. branchial arch stages;cartilage and bone formation;palatal shelf formation) to test if early knockdown leads to later problems in migration and/or formation of facial derivatives. Taking together we will systematically address the epigenetic role of PHD12 on NC EMT and it implication on the formation of craniofacial derivatives.

Public Health Relevance

Most of the craniofacial skeleton arises from the neural crest (NC), a multipotent stem-cell-like population that migrates extensively and gives rise to an amazingly diverse set of derivatives. In addition to specific components of the craniofacial skeleton, neural crest cells also form melanocytes, neurons and glia of the peripheral nervous system, and smooth muscle. Moreover, many craniofacial abnormalities have their origin in problems at different phase of neural crest cell development ranging from initial formation, to migration or differentiation. One major contributor to craniofacial defects occurs during their departure from the neural tube, which involves a classic epithelial-to-mesenchymal transition (EMT). This application examines epigenetic role of PHD12 in NC EMT and its long term effects on formation and development of the face. Because neural crest-derived cells are involved in a variety of birth defects such as cleft palate and various types of cancer, our results on the normal mechanisms of neural crest development will provide important clues regarding the mistakes that may lead to abnormal development or loss of the differentiated state.